Landscapes of Enteric Virome Signatures in Early-Weaned Piglets

ABSTRACT Diarrhea caused by early-weaning-induced stress can increase mortality rates and reduce growth performance of piglets, seriously harming the livestock industry. To date, studies on the gut microbiome of early-weaned piglets have focused almost exclusively on bacteria, while studies on their gut virome are extremely lacking. Here, we used metagenomic and metatranscriptomic sequencing combined with bioinformatic analysis techniques to preliminarily characterize the intestinal virome of early-weaned piglets at different biological classification levels. The alpha diversity of enteroviruses was generally elevated in early-weaned piglets with diarrhea, compared to healthy piglets, whereas the two groups of piglets showed no significant difference in beta diversity. In addition, the species compositions of the gut virome were similar between healthy piglets and piglets with diarrhea, while their respective dominant species were somewhat different. We also identified 58 differential DNA viruses and 16 differential RNA viruses between the two groups of piglets at all biological taxonomic levels. Of these, 1 (family Dhakavirus) and 6 (phylum Artverviricota, class Revtraviricetes, order Ortervirales, family Retroviridae, genus Gammaretrovirus, and species Kirsten murine sarcoma virus) specific viruses disappeared from the intestines of healthy piglets and piglets with diarrhea, respectively. Moreover, we found that some DNA and RNA viruses formed strong correlations among themselves or between them. IMPORTANCE This study systematically reveals the biological diversity, structure, and composition of intestinal DNA and RNA virus profiles in early-weaned piglets. Furthermore, characteristics of differences in gut viromes between early-weaned healthy piglets and piglets with diarrhea were also elucidated. Importantly, some potential biomarkers for early-weaned piglets with diarrhea were identified. These findings fill a gap for the early-weaned piglet gut virome and lay the foundation for the development of strategies to target enteroviruses for the prevention and treatment of early-weaning-induced piglet diarrhea.

metagenomics and metatranscriptomics. DNA-and RNA-viruses are analyzed by sequencing of total DNA and total RNA present in the fecal samples of 12 healthy-and 12 diarrheic early-weaned piglets. Based on this analysis, authors report that the alpha-diversity is elevated in the diarrheic piglets but no significant change is observed in the beta-diversity. 8 viruses are identified as differentially present species in the two populations. Also, that this is a first report on comparison of virome composition in healthy and diarrheic early-weaned piglets.
I have following comments-Major Points: 1. Authors have mentioned that their dataset has been submitted to NCBI SRA repository with accession number PRJNA775062. However, it is not accessible.
2. Authors have reported that two viruses, Dhakavirus and Lactobacillus prophage Lj928 are absent in the healthy (earlyweaned) piglets and that six viruses are absent in the diarrheic piglets. However, to be able to consider that these differences are statistically significant, the sample size (12 in each population) used in this study is not sufficient. PCR method could be used to detect the 8 viruses in question in larger sample size to establish the significance.
3. Line 358-359: contigs longer than 10, 000 bp were retained for DNA and the threshold for RNA reads was 2000 bp. The reason for these cut-offs should be mentioned because many DNA virus genomes are shorter than 10, 000 bp. 4. Line 361: Why were VirSorter categories, 3 & 6 excluded? 5. Line 366: What is meant by "viral catalog"? 6. What database(s) is used for taxonomic annotation?
Minor Points: The authors have called their study population as "health piglets" and "diarrhea piglets" at some places whereas it is mentioned as "healthy piglets" and "diarrheic piglets" at some. Please correct. Line 76-77: Gut virome cannot be called a technology. Line 105: Figure "1A-C and E" should be "1A, C and E". Line 111: "Different at the phylum, class, order and genus" should be changed to "Different at the phylum, class, order and family". Line 113: "at the phylum, order, family" should be "at the phylum, class, order, family". Line 199: Please clarify if the co-occurrence analysis is done in healthy, diarrheic or both groups of piglets. Also, is there a rationale to analyze co-occurrence of DNA and RNA viruses? Line 583: "Species composition of intestinal", should be changed to "Composition of intestinal" because the figure is showing composition at levels other than Species too.
Reviewer #2 (Public repository details (Required)): Metagenomics and metatranscriptomics data were generated for this study, so they need to be deposited Reviewer #2 (Comments for the Author): Major comments: There are DNA viruses listed as RNA viruses, because they were detected using metatranscriptomics (Supplementary table 2 , etc). Can the authors clarify throughput the manuscript that some DNA viruses were detected using metaranscriptomics means that these were actively transcribing genes? For example, in lines 186 and 241, were these RNA viruses based on their genomes, or some of these were DNA viruses that were identified using the metatranscriptomics pipeline and thus were grouped with the RNA viromes? This seems to be the case for some of the Lactobacillus phages and prophages, shown in figure 8, which are DNA viruses, but were captured using metatranscriptomics.
Did the authors look into correlations between the DNA viromes, and the bacteriome? This may be important to discuss and show any bacteria-virus interactions, similar to those shown in Figure 9 between the DNA and RNA viromes. Figure 1 shows important information regarding alpha diversity, but the figure may be too crowded and deviate from the main message. The figure may benefit from just showing the plots with significant differences, including panels D and F. Alternatively, panels D, E and F can be shown. Panels A, B, and C can be mentioned in the text, or moved to supplementary material. Same with Figure 2. Alternatively, alpha diversity indices for DNA and metatranscriptomics can be shown in the same figure for both healthy and piglets with diarrhea. This may facilitate comparing the alpha diversity values for both the DNA and RNA viromes in one plot.
The authors mentioned a package called Salmon. Can they further explain how abundances were obtained using this package and if the relative abundances were obtained by dividing the number of contigs by the total number of contigs?
Other comments: Lines 174-176: Are these numbers respective to the taxonomic levels? Please clarify.
Can the authors clarify the number of reads or sequencing depth used in the analyses? This is important to include as sequencing depth is known to affect virome results.

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Weaning of piglets at 21 days after birth is considered early-weaning but it is practiced in pig farms, in order to improve farming efficiency. It has been reported that early-weaned piglets are at risk for intestinal dysfunction, which can lead to diarrhea and even death. In this study, authors have explored if gut virome is associated with diarrhea in early-weaned piglets. They have performed analysis of DNA-and RNA-viruses in diarrheic-and healthy early-weaned piglets, through metagenomics and metatranscriptomics. Total DNA and total RNA, present in the fecal samples of 12 healthy-and 12 diarrheic early-weaned piglets are sequenced and analyzed in this study. Based on this analysis, authors report that (i) the α-diversity is elevated in the diarrheic piglets but no significant change is observed in their β-diversity; (ii) 8 viruses are identified as differentially present species in the two populations; (iii) this is a first report on comparison of virome composition in healthy and diarrheic early-weaned piglets.
I have following comments-Major Points: 1. Authors have mentioned that their dataset has been submitted to NCBI SRA repository with accession number PRJNA775062. However, it is not accessible.
2. It would be useful to provide Read and Contig Summary for each sample as a suppl. Table. 3. Authors have reported that two viruses, Dhakavirus and Lactobacillus prophage Lj928 are absent in the healthy (early-weaned) piglets and that six viruses are absent in the diarrheic piglets. However, to be able to consider that these differences are statistically significant, the sample size (12 in each population) used in this study is not sufficient. PCR method could be used to detect the 8 viruses in question in larger sample size to establish the significance.
4. Line 358-359: contigs longer than 10, 000 bp were retained for DNA and the threshold for RNA reads was 2000 bp. The reason for these cut-offs should be mentioned because many DNA virus genomes are shorter than 10, 000 bp.

Minor Points:
Authors have called their study populations as "health piglets" and "diarrhea piglets", at some places whereas they are mentioned as "healthy piglets" and "diarrheic piglets" at some. Please correct.
It would be helpful to mention the extent of early-weaning practice and how it is being managed presently.
What kind of associations have been reported between early-weaning and microbiome of piglets, in earlier studies?
Line 76-77: Gut virome cannot be called a technology. Line 105: Figure "1A-C and E" should be "1A, C and E". Line 111: "Different at the phylum, class, order and genus" should be changed to "Different at the phylum, class, order and family". Line 113: "at the phylum, order, family" should be "at the phylum, class, order, family". Line 199: Please clarify if the co-occurrence analysis is done in healthy, diarrheic or both groups of piglets. Also, is there a rationale to analyze co-occurrence of DNA and RNA viruses? Line 583: "Species composition of intestinal", should be changed to "Composition of intestinal" because the figure is showing composition at levels other than Species too.

Reviewer #1 (Public repository details (Required)):
Authors have mentioned that the Dataset has been submitted to NCBI SRA repository and accession number is provided (PRJNA775062). However, it is not accessible.

Reply:
We have changed the opening times of the dataset which is now accessible.

Reviewer #1 (Comments for the Author):
In this study, authors have performed analysis of the intestinal virome in diarrheicand healthy early-weaned piglets, through metagenomics and metatranscriptomics.
DNA-and RNA-viruses are analyzed by sequencing of total DNA and total RNA present in the fecal samples of 12 healthy-and 12 diarrheic early-weaned piglets.
Based on this analysis, authors report that the alpha-diversity is elevated in the diarrheic piglets but no significant change is observed in the beta-diversity. 8 viruses are identified as differentially present species in the two populations. Also, that this is a first report on comparison of virome composition in healthy and diarrheic early-weaned piglets.
I have following comments-Major Points: 1. Authors have mentioned that their dataset has been submitted to NCBI SRA repository with accession number PRJNA775062. However, it is not accessible.

Reply:
We have changed the opening times of the dataset which is now accessible.

Authors have reported that two viruses, Dhakavirus and Lactobacillus prophage
Lj928 are absent in the healthy (early-weaned) piglets and that six viruses are absent in the diarrheic piglets. However, to be able to consider that these differences are statistically significant, the sample size (12 in each population) used in this study is not sufficient. PCR method could be used to detect the 8 viruses in question in larger sample size to establish the significance.
Reply: Thank you very much for your professional suggestion. Based on your suggestion, we tried hard to collect more samples, but since the situation of African Swine Fever in China is still very serious and pig farms are under closed management, we could not get more samples.
In fact, like all other differential viruses, these viruses were also differential viruses identified using the statistical method of Wilcoxon Rank-Sum Test. In addition, a number of reported literatures have demonstrated that 12 biological replicates per group are sufficient to be statistically significant.
The authors have called their study population as "health piglets" and "diarrhea piglets" at some places whereas it is mentioned as "healthy piglets" and "diarrheic piglets" at some. Please correct.

Reply:
We have unified "health piglets" and "diarrhea piglets" in the manuscript.
Line 76-77: Gut virome cannot be called a technology.
Line 111: "Different at the phylum, class, order and genus" should be changed to "Different at the phylum, class, order and family".
Line 113: "at the phylum, order, family" should be "at the phylum, class, order, family".

Reply: Revised.
Line 199: Please clarify if the co-occurrence analysis is done in healthy, diarrheic or both groups of piglets. Also, is there a rationale to analyze co-occurrence of DNA and

RNA viruses?
Reply: What we used for co-occurrence analysis were differential DNA and RNA viruses between health and diarrhea piglets. At present, the potential link between DNA and RNA viruses has not been well revealed, and our study conducted a Spearman correlation analysis based on differential DNA and RNA viruses to initially suggest a possible link between them.
Line 583: "Species composition of intestinal", should be changed to "Composition of intestinal" because the figure is showing composition at levels other than Species too.

Reviewer #2 (Public repository details (Required)):
Metagenomics and metatranscriptomics data were generated for this study, so they need to be deposited We have changed the opening times of the dataset which is now accessible.

Reviewer #2 (Comments for the Author):
Major comments: There are DNA viruses listed as RNA viruses, because they were detected using metatranscriptomics (Supplementary Did the authors look into correlations between the DNA viromes, and the bacteriome?
This may be important to discuss and show any bacteria-virus interactions, similar to those shown in Figure 9 between the DNA and RNA viromes.
Reply: Thank you very much for your professional suggestion. The correlation analysis between bacteria and DNA viruses is indeed beneficial to show the bacteria-virus interactions. In this study, we focused on the intestinal viral characteristics of health and diarrhea piglets. In addition, we have submitted metagenomics sequencing data (bacteria) to other journals, in order to strictly follow academic ethics and avoid conflicts of interest, so we did not involve any information concerning bacteria in this study. We hope to have your understanding. Reply: Thank you very much for your professional suggestion. We believe that even though some of the results are not significantly different, they are still important information in our study. In order to avoid overcrowding of the figures while preserving all the results information, we recombined the content in Figures 1 and 2.
The authors mentioned a package called Salmon. Can they further explain how abundances were obtained using this package and if the relative abundances were obtained by dividing the number of contigs by the total number of contigs?
Reply: The relative abundances of viral genomes were calculated by Salmon within MetaWRAP package which was widely used in metagenomic analysis. In gene abundance calculation, Salmon not only considers the number of contigs and reads, but also considers the GC bias and length of reads. And the detailed algorithm for Salmon can be tracked in the software website (https://salmon.readthedocs.io/en/latest/).